Article
assay medium was added to each well and plates were equilibrated at
37 °C for 1 h. Four measurements of basal oxygen consumption rates
(picomoles per minute) were recorded on a Seahorse Bioscience XFe
24 Analyzer (Seahorse Biosciences) using an instrument protocol of
3-min mix, 2-min wait and 3-min measure. After baseline measurements,
glucagon (or vehicle) was injected at a final concentration of 100 nM
and oxygen consumption was recorded using the same instrument
protocol. Ten measurements were taken following injection and the
average of eight measurements was used for subsequent analyses.
Oxygen consumption rates were normalized to total protein content
and expressed as fold change compared to vehicle-treated cells.
Statistics and reproducibility
Group sizes were chosen to detect moderate-to-large (>40%) differ-
ences with approximately 40% standard deviations. Comparisons were
performed using GraphPad Prism 7. Each in vivo experiment was per-
formed with the number of replicates specified in the figure legends.
All data obtained are shown, with the exception of several western blots
in which case one representative image is shown; all data are shown in
the quantification. The two-tailed paired (when comparing the same
animals under multiple conditions) or unpaired Student’s t-test (when
comparing different animals) was used to compare datasets. P values
less than 0.05 were considered significant. Comparisons were per-
formed using GraphPad Prism 7.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
All data generated and analysed in this study are available in the Article
and its Supplementary Information files, which include a table contain-
ing all raw data. Source Data for Figs. 1–3, Extended Data Figs. 1–8 are
included with the paper.
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Acknowledgements We thank M. Montminy, T. Sonntag and Y.-S. Yoon for kindly providing
the CRTC2 antibody and control samples, and for their advice on the interpretation of the
CRTC2 blots; H. S. Sui for generously providing the pATGL antibody; D. Vatner for his advice on
these studies; and X. Ma, J. Dong, W. Zhu, M. Kahn, K. Harry and M. Batsu for their expert
technical assistance. These studies were funded by grants from the United States Public
Health Service (R01 DK113984, P30 DK059635, P30 DK034989, T32 DK101019, K99/R00
CA215315, R01 NS087568, UL1TR000142, T32 DK007058 and F32 DK114954). The content is
solely the responsibility of the authors and does not necessarily represent the official views of
the NIH.Author contributions The study was designed by R.J.P. and G.I.S. Data were collected and
analysed by R.J.P., D.Z., M.T.G., A.L.B., L.G., A.R.N., A.R.-C., Y.W., L.P., S.D., Y.Z., X.-M.Z., G.M.B.,
K.T., Y.N., K.F.P., G.W.C., B.E.E. and M.H.N. The manuscript was written by R.J.P. and G.I.S. with
contributions and approval from all authors.
Competing interests The authors declare no competing interests.Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
2074-6.
Correspondence and requests for materials should be addressed to G.I.S.
Peer review informationNature thanks Ilya Bezprozvanny and the other, anonymous,
reviewer(s) for their contribution to the peer review of this work.
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